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Authentication markers for five major Panax species developed via comparative analysis of complete chloroplast genome sequences Van Binh Nguyen, Hyun-Seung Park, Sang-Choon Lee, Junki Lee, Jee Young Park, and Tae-Jin Yang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 22 May 2017 Downloaded from http://pubs.acs.org on May 23, 2017

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Journal of Agricultural and Food Chemistry

Authentication markers for five major Panax species developed via comparative analysis of complete chloroplast genome sequences

Van Binh Nguyena, Hyun-Seung Parka, Sang-Choon Leea, Junki Leea, Jee Young Parka, TaeJin Yanga,b* a

Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute

of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Republic of Korea. b

Crop Biotechnology Institute/GreenBio Science and Technology, Seoul National University,

Pyeongchang 232-916, Republic of Korea. *Corresponding author: Tae-Jin Yang E-mail: [email protected] Tel: +82-2-880-4547, Fax: +82-2-877-4550

1 ACS Paragon Plus Environment


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Abstract

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Ginseng represents a set of high-value medicinal plants of different species: Panax

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ginseng (Asian ginseng), P. quinquefolius (American ginseng), P. notoginseng (Chinese

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ginseng), P. japonicus (Bamboo ginseng), and P. vietnamensis (Vietnamese ginseng). Each

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species is pharmacologically and economically important, with differences in efficacy and

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price. Accordingly, an authentication system is needed to combat economically motivated

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adulteration of Panax products. We conducted comparative analysis of the chloroplast

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genome sequences of these five species, identifying 34–124 InDels and 141–560 SNPs.

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Fourteen InDel markers were developed to authenticate the Panax species. Among these,

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eight were species-unique markers that successfully differentiated one species from the others.

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We generated at least one species-unique marker for each of the five species, and any of the

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species can be authenticated by selection among these markers. The markers are reliable,

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easily detectable, and valuable for applications in the ginseng industry as well as in related

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research.

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Keywords: Panax species, Chloroplast genome, Molecular markers, Ginseng authentication


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Introduction

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The Panax genus belongs to the Araliaceae family and contains many important

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medicinal species, collectively called ‘ginseng’. Of the 14 known species in the Panax genus,

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five species, Panax ginseng (Asian ginseng), P. quinquefolius (American ginseng), P.

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notoginseng (Sanchi ginseng; Chinese ginseng), P. japonicus (Bamboo ginseng), and P.

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vietnamensis (Vietnamese ginseng), are broadly utilized in Korea, the USA, China, Japan,

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and Vietnam. Each species is well-known as a traditional medicinal plant in oriental countries,

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and species such as P. ginseng, P. quinquefolius, and P. notoginseng contain

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protopanaxadiol-type and protopanaxatriol-type saponins1, while other species like P.

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japonicus and P. vietnamensis contain high quantities of oleanolic acid-type and ocotillol-

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type saponins, respectively1,2.

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The high pharmacological and economical value of ginseng means that many

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economically motivated adulterations (EMAs) of ginseng products have been developed by

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substituting morphologically similar plant roots, or by mixing different species. Traditionally,

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the authentication of herb plants was based on morphological and histological inspection.

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However, these traditional methods are unable to authenticate some Panax species because of

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their very similar morphological appearances, especially in terms of root shape. For example,

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P. ginseng and P. quinquefolius, and P. japonicus and P. vietnamensis cannot easily be

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distinguished from each other. Moreover, many commercial ginseng products are sold in a

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processed form, such as red ginseng, ginseng powder, liquid extracts, pellets, shredded slices,

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or even tea, which cannot be authenticated by traditional methods. Ginsenoside profiling

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methods have been developed to authenticate ginseng samples3. However, the effects of

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factors such as growth conditions, developmental stage, internal metabolism, storage

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conditions, and manufacturing processes on secondary metabolite accumulation in ginseng

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limits the application of such chemical analyses4. Chemical methods are also expensive and



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difficult to utilize in high-throughput analysis. Therefore, reliable and practical methods to

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authenticate ginseng are in high demand.

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The chloroplast is a plant-specific organelle containing the entire machinery required for

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photosynthesis and carbon fixation. Chloroplast genomes are generally highly conserved

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across land plants at the gene level, with a quadripartite structure comprising two inverted

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repeat blocks (IRs), one large single copy (LSC) region, and one small single copy (SSC)

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region. As a result of interspecies sequence divergence and intraspecies sequence

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conservation, the chloroplast genome is valuable for taxonomic classification and phylogeny

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reconstruction5,6. Lack of recombination, low nucleotide substitution rates, and usually

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uniparental inheritance make chloroplast genomes valuable sources of genetic markers for

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phylogenetic analysis and species identification7,8. Chloroplast sequences such as atpF-atpH,

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matK, psbK-psbI, rbcL, ropC1, rpoB, and trnH-psbA are commonly used as DNA barcodes

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for plants9-11. In some cases, these sequences were highly efficient for species identification

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and phylogenetic studies, but they showed low variation in closely related species10.

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Recently, DNA markers have been developed to authenticate ginseng, including random

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amplified polymorphic DNA (RAPD)12, microsatellite13, and expressed sequence tag-simple

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sequence repeat (EST-SSR)14,15 markers, as well as single nucleotide polymorphism (SNP)

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and insertion and deletion (InDel) markers derived from chloroplast sequences6,16,17.

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However, thus far, these markers have been used to identify only P. ginseng cultivars at the

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intraspecies level, and just a few Panax species at the interspecies level.

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Recently, we developed an efficient method to obtain complete chloroplast genome and

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nuclear ribosomal DNA (nrDNA) by de novo assembly using low-coverage whole-genome

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shotgun next-generation sequencing (dnaLCW)18. Using this method, we obtained complete

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chloroplast genomes and nrDNA for the five Panax species: P. ginseng, P. quinquefolius, P.

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notoginseng, P. japonicus, and P. vietnamensis6,19. In the present study, we comparatively


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analyzed these five chloroplast genome sequences and developed credible chloroplast

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genome-derived InDel markers to authenticate these Panax species. These markers are

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valuable tools for the further study of genetic diversity in the Panax genus. They also may be

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used to support the ginseng industry, which depends on a number of Panax species, for

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example, P. ginseng in Korea, China, and Japan, P. quinquefolius in the USA, Canada, and

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China, P. notoginseng in China, and P. vietnamensis in Vietnam.

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Materials and Methods

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Plant materials and Genomic DNA extraction

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P. ginseng (cultivars ‘Chunpoong’ (CP) and ‘Yunpoong’ (YP)) and P. quinquefolius

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plants were collected from the ginseng farm at Seoul National University in Suwon, Korea. P.

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notoginseng and P. japonicus plants were collected from Dafang County, Guizhou Province,

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and Enshi County, Hubei Province, China, respectively. P. vietnamensis plants were collected

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from Ngoc Linh Mountain, Kon Tum Province, Vietnam. Individual leaves and roots of

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plants from each species were harvested and stored at −70°C until use. Total genomic DNA

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was isolated using a modified cetyltrimethylammonium bromide (CTAB) method20. The

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quality and quantity of extracted genomic DNA was measured using a UV-spectrophotometer.

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Comparative analysis and characterization of SSRs and large sequence repeats

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The chloroplast genome sequences of P. ginseng cv. CP (KM088019), P. ginseng cv. YP

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(KM088020), P. quinquefolius (KM088018), P. notoginseng (KP036468), P. japonicus

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(KP036469), and P. vietnamensis (KP036471) were obtained from our previous studies6,19.

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MAFFT

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(http://genome.lbl.gov/vista/mvista/submit.shtml) programs were used to compare these

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sequences.

(http://mafft.cbrc.jp/alignment/server/),

MEGA


621,

and

mVISTA


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SSRs were predicted using MISA (http://pgrc.ipk-gatersleben.de/misa/misa.html)22 with

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the parameters set to ≥10 repeat units for mononucleotide SSRs, ≥5 repeat units for

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dinucleotide SSRs, ≥4 repeat units for trinucleotide SSRs, and ≥3 repeat units for

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tetranucleotide, pentanucleotide, and hexanucleotide SSRs.

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The program REPuter23 was used to identify the number and location of repeat sequences,

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including tandem, dispersed, reverse, and palindromic repeats within chloroplast genomes.

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For all repeat types, the following constraints were set to a minimum repeat size of 15 bp, and

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90% minimum cut-off identity between two copies. Overlapping repeats were merged into

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one repeat motif whenever possible.

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Development and validation of InDel markers

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To validate intraspecies and interspecies polymorphism in the chloroplast genomes, and

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develop DNA markers to authenticate the five major Panax species, specific primers were

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designed based on InDel polymorphic sites found in Panax chloroplast genomes, including P.

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ginseng cv. YP (KM088020) as an intraspecies level. Primer pairs were designed using the

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Primer3 program (http://bioinfo.ut.ee/primer3-0.4.0/).

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The polymerase chain reaction (PCR) was performed in a 25 µl reaction mixture

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containing 20 ng of DNA template, 5 pmol of each primer, 1.25 mM deoxynucleotide

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triphosphate (dNTP), 1.25 units of Taq DNA polymerase (Inclone, Korea), and 2.5 µl of 10×

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reaction buffer. The PCR reaction was performed in thermocyclers using the following

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cycling parameters: 94°C (5 min); 35 cycles of 94°C (30 s), 54–58°C (30 s); 72°C (30 s),

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then 72°C (7 min). PCR products were visualized on agarose gels (1.5–3.0%) after staining

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with ethidium bromide, and/or analyzed by capillary electrophoresis using a fragment

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analyzer (Advanced Analytical Technologies Inc., USA).

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Results


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Structure of complete chloroplast genomes of five Panax species

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Complete chloroplast genomes of five Panax species were obtained by dnaLCW and

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reported in our previous studies6,19. Lengths of the chloroplast genomes ranged from 155,993

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bp (P. vietnamensis) to 156,466 bp (P. notoginseng). The order, content, and orientation of

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genes were highly conserved among these five chloroplast genomes, comprising 79 protein-

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coding genes, 30 tRNA genes, and 4 rRNA genes in common. Each chloroplast genome had

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the same quadripartite structure with an LSC, an SSC, and two IR regions (Fig. 1).

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Variations in the copy numbers of SSRs were identified among the five Panax chloroplast

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genomes. The longest SSRs were 18 nucleotides in length, and the most abundant nucleotides

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in SSRs were A and T (Table 1). P. vietnamenis and P. notoginseng both contained 42 SSRs;

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lower than P. japonicus (45), and higher than P. ginseng cv. CP (38) and P. quinquefolius (40)

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(Table 1). The P. japonicus chloroplast genome has the highest number of homopolymers

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(24), but no hexapolymers. P. vietnamensis, P. notoginseng, and P. ginseng cv. CP all had 6

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dipolymers; lower than P. japonicus and P. quinquefolius (7). P. notoginseng had 4

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tripolymers; more than each of the other Panax species (3). P. vietnamensis had 10

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tetrapolymers; more than the other Panax species (8). P. japonicus and P. notoginseng had 3

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pentapolymers each, while the other Panax species contained 2 (Table 1).

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For comparative analysis, repetitive sequences were grouped into four categories: tandem,

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dispersed, palindromic, and reverse. To avoid redundancy, repeat sequence analysis of each

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chloroplast genome was carried out with a single IR region. A total of 45–50 repetitive

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sequences were identified in each of the five Panax chloroplast genomes (Fig. 2B), including

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dispersed repeats (44.26%), palindromic repeats (29.79%), tandem repeats (22.13%), and

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reverse repeats (3.83%). Most repeats were located in intergenic spaces (IGS) (53.83%) and

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coding sequence (CDS) regions (38.94%); a small number of repeats (7.23%) were found in

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intron regions (Fig. 2C). Across the five chloroplast genomes, repeat lengths ranged from



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17 bp to 67 bp (Fig. 2A). The 67-bp tandem repeat found in P. japonicus was the longest

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repeat (Fig. 2A). Palindromic and reverse repeats ranged from 17–35 bp and 17–28 bp,

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respectively (Fig. 2A). Comparing the length and location, 32 repeats were shared by all five

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Panax species, and 5 repeats were present in four chloroplast genomes. P. notoginseng had

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the most unique repeats (9), followed by P. ginseng and P. vietnamensis (6), P. japonicus (3),

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and P. quinquefolius (1) (Fig. 2D).

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Divergence of chloroplast genomes among Panax species

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To investigate chloroplast genome divergence among the five Panax species, multiple

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alignments of six chloroplast genomes were performed with a single IR region. The sequence

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identity of six chloroplast genomes was plotted using the mVISTA program, using the Panax

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ginseng cv. CP annotation as the reference (Fig. 3). Two cultivating varieties of ginseng, CP

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and YP, were almost identical with 3 InDels and 1 SNP overall (Table 2). Five Panax species

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also showed high sequence similarity with each other (98.9%) (Fig. 3), suggesting that Panax

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chloroplast genomes are well conserved. Our results are consistent with earlier research on

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Araliaceae chloroplast genomes8. As expected, coding regions are more conserved than non-

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coding regions. The most highly conserved chloroplast genome coding regions are the four

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rRNA and 30 tRNA genes (Fig. 3). The most divergent coding region is the ycf1 gene, which

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has low sequence identity because of various InDels and repeat sequences (Fig. 3). This has

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been reported in previous research on other chloroplast genomes8,24.

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Although chloroplast genome diversity at the intraspecies level is low, abundant

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polymorphism was identified between species. At the interspecies level, the number of SNPs

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ranged between 141 (P. ginseng versus P. quinquefolius) and 560 (P. ginseng versus P.

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vietnamensis), and the number of InDels ranged between 34 (P. ginseng versus P.

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quinquefolius) and 124 (P. notoginseng versus P. vietnamensis) (Table 2). A/T SNP

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substitutions were more frequent than other types, in agreement with previous studies5,25.


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Fewer substitutions and InDels were found between P. ginseng and P. quinquefolius than

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between the other three Panax species. The ratios of nucleotide substitution events to InDel

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events (S/I) for different pairwise comparisons between species ranged between 4.06 (P.

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ginseng versus P. quinquefolius) and 5.64 (P. quinquefolius versus P. japonicus) (Table 2).

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S/I ratios are thought to increase with divergence time between genomes26. Our results

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indicate that P. ginseng is more closely related to P. quinquefolius (S/I = 4.06), and that P.

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quinquefolius is more highly divergent from P. japonicus (S/I = 5.64), compared to others.

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Development of InDel markers to identify Panax species

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Based on chloroplast genome sequence alignment, the 14 most InDel-variable loci were

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selected to develop 14 potentially discriminate markers (Table 3; Fig. 1). Each of these 14

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markers were successfully amplified by PCR, and each showed the expected polymorphic

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band sizes. Eight of these 14 markers had unique amplicon sizes specific to different Panax

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species (Table 4).

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The marker gcpm2 was specific to both P. ginseng cultivars (CP and YP), and was

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derived from a 33-bp tandem repeat (TR) in the rps16–trnQ-UUG region (Table 4; Fig. 4B).

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The P. notoginseng-specific markers gcpm3, gcpm8, and gcpm10 were derived from a 25-bp

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TR in the atpH–atpI region, a 38-bp TR in the petA–psbJ region, and a 25-bp TR in the

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rpl14–rpl16 region, respectively (Table 4; Fig. 4C, H, K). The gcpm4 marker was derived

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from a 23-bp TR in the rps2–rpoC2 region, and was specific to P. quinquefolius (Table 4; Fig.

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4D). The marker gcpm6 was derived from a 67-bp TR and a 19-bp InDel in the trnE–trnT

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region, and was specific to P. japonicus (Table 4; Fig. 4F). Finally, the markers gcpm9 and

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gcpm14 were derived from a 25-bp TR and a 6-bp SSR in the clpP–psbB region, and a 30-bp

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InDel in ycf1, respectively, and were specific to P. vietnamensis (Table 4; Fig. 4I, O).

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Validation results revealed that six markers, gcpm1, 5, 7, 11, 12, and 13, were able to

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identify more than two species, of which gcpm12 (derived from a 57-bp TR in the ycf1 gene)



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was the most variable (Table 4; Fig. 4M). In addition, gcpm12 also distinguished between the

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two P. ginseng cultivars (Table 4; Fig. 4M).

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Discussion

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Repetitive sequences in Panax chloroplast genomes

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Repeat structure plays an important role in genomic rearrangement and divergence in

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chloroplast genomes via illegitimate recombination and slipped-strand mispairing27,28. SSRs

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are direct tandem repeated DNA sequences consisting of short (1–10 bp) nucleotide motifs.

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The highly polymorphic, non-recombinant and uniparentally inherited nature of SSRs means

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they are often used as genetic molecular markers for population genetics29,30, and the study of

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ecology and evolution. In this study, we found 207 SSRs (Table 1), varying in number and

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type between five major Panax species.

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It is considered that species divergence in chloroplast genomes is mostly caused by

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repetitive sequences. Indeed, we found many repetitive sequences associated with

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polymorphic chloroplast sites among the five major Panax species studied. Among the many

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repeats in the ycf3 gene, the most divergent target region was a 57-bp TR. TRs such as this

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can be used to develop molecular markers for the identification and authentication of Panax

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species6.

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Comparative analysis of Panax chloroplast genomes

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Polymorphism at the intraspecies level polymorphism is very low compared to that at the

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interspecies level. In P. ginseng, 12 chloroplast genomes derived from different cultivating

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varieties revealed over 99.9% sequence similarity, and only 6 InDels and 6 SNPs6.

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Meanwhile, of a total of 201 InDels and 962 SNPs identified among five Panax chloroplast

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genome sequences, 34–124 InDels and 141–560 SNPs were shared between two Panax


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species (Table 2). However, the chloroplast genome is highly conserved within Panax species,

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and has high similarity (≥98.9%) at the nucleotide sequence level.

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In chloroplast genomes, nucleotide substitution has been used to examine plant evolution

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and genome differentiation between species7,17. These InDel events are mainly attributed to

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the repetition of an adjacent sequence, probably resulting from slipped-strand mispairing in

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DNA replication31. InDels play a major role in genome size evolution and are increasingly

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used in phylogenetic studies32,33. S/I ratios have been reported to increase with divergence

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time between genomes26. In this study, we identified many SNPs and InDels from the

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complete chloroplast genomes of five major Panax species; S/I ratios ranged between 4.06 (P.

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ginseng versus P. quinquefolius) and 5.64 (P. quinquefolius versus P. japonicus) (Table 2).

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Along with their similar morphologies and distributions, this indicates that P. ginseng is

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closely related to P. quinquefolius (S/I = 4.06), and is consistent with previous reports34,35. P.

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quinquefolius is highly divergent from P. japonicus compared to others (S/I = 5.64).

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Use of molecular markers to authenticate ginseng species

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Biological diversity is a valuable and vulnerable natural resource. The first steps towards

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protecting and benefiting from national biodiversity are to sample, identify, and study

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biological specimens. The use of molecular markers (i.e., DNA barcoding) has a powerful

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role to play in attaining many of the Millennium Development Goals, and reaching the

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objectives of the Convention on Biological Diversity, by sustaining natural resources,

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protecting endangered species, and identifying pests and pathogens at any life stage so as to

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more easily control them. DNA barcoding can also help to monitor food, water and

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environment quality by studying contaminating organisms.

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Although relatively new, the use of molecular markers is well on its way to being

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accepted as a global standard for species identification, and will play a major role in the

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future of taxonomy36. DNA-based species identification offers enormous potential for the



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biological scientific community, educators, and the interested public. The complete

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chloroplast genome has a conserved sequence from 110–160 kbp, which far exceeds the

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length of commonly used molecular markers and provides greater variation to discriminate

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between closely related species37. The chloroplast genome is smaller in size and hundreds

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times of higher copy numbers in a cell compared with the nuclear genome and has solid

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interspecific divergence with lower intraspecific variation, which makes chloroplast genome-

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based marker is suitable as DNA barcoding target37.

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It is important to be able to authenticate natural health products (NHPs) from legal,

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economic, health and conservation viewpoints. NHPs are often trusted by the public to be

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safe; however, adulterated, counterfeit and low quality products can seriously threaten

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consumer safety38,39. Ginseng plants have high economic and medicinal value, thus there are

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many EMA ginseng products. The use of molecular methods to accurately identify the origins

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of ginseng products is important to the development of the ginseng industry in those countries

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where ginseng is cultivated, such as Korea, the USA, China, Japan, and Vietnam. Recently,

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the chloroplast genome sequences rbcL, matK, trnH-psbA, and trnL-trnF, and a nuclear

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internal transcribed spacer (ITS) of nuclear 45S ribosomal RNA genes, have successfully

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been used as molecular markers for several plant species9,40, but these loci have little

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variability in Panax36. Although many studies have sought to authenticate ginseng13-15,41,

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application remains limited because of a lack of genome information and comprehensive

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comparative genomic analysis against related Panax species.

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In this study, we developed chloroplast-derived, species-specific markers for each of five

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major Panax species. Among 14 molecular markers, three markers were specific to P.

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notoginseng, and two were specific to P. vietnamensis. P. ginseng, P. quinquefolius and P.

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japonicus could also each be identified by species-specific markers (Table 4; Fig. 4). We

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discovered different 57-bp tandem repeats in the ycf1 gene of different Panax chloroplast


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genomes, and this polymorphism proved powerful in the identification of Panax species

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(Table 4; Fig. 4M). However, the gcpm12 marker, derived from the ycf1 gene, unexpectedly

266

produced two amplified bands from P. notoginseng, of which the A allele was the expected

267

allele of the P. ginseng type; allele D was not expected, but was same as the allele of P.

268

Vietnamese, although all other species gave rise to a single band (Table 4; Fig. 4A, M). We

269

assume that these unexpected bands are derived from heteroplasmy of the target sequences

270

caused by different IRA and IRB regions related to ycf1 gene sequences; alternatively, they

271

could be caused by chloroplast DNAs transferred into the nuclear or mitochondrial

272

genomes42-45. In our previous study6, we also found intra-species polymorphism in P. ginseng

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at this position, indicating that, although highly informative, the marker designed from this

274

region may be confusing for species authentication. Overall, we suggest that intra-species

275

polymorphism and a combination of several markers should be considered for credible

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authentication between different species.

277 278

Abbreviation Used

279

InDel, insertion or deletion; SNP, single nucleotide polymorphism; DNA, deoxyribonucleic

280

acid; bp, base pair.

281 282

Funding

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This work was supported by: the Cooperative Research Program for Agriculture Science &

284

Technology Development of the Rural Development Administration (grant number

285

PJ01100801); the Ministry of Food and Drug Safety (grant number 16172MFDS229,

286

awarded in 2016); and the Bio & Medical Technology Development Program of the NRF,

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MSIP (grant number NRF-2015M3A9A5030733), Republic of Korea.

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Chen, S. L.; Yang, J. B. Comparative analysis of a large dataset indicates that internal

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Baeshen, N. A.; Jansen, R. K. Complete sequences of organelle genomes from the

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110.

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DNA flux. The Plant Cell. 2005, 17, 665-675.

419

420



421

FIGURE CAPTIONS

422

Fig. 1. Complete chloroplast genomes of five Panax species.

423

Colored boxes show conserved chloroplast genes, classified based on product function.

424

Genes shown inside the circle are transcribed clockwise, and those outside the circle are

425

transcribed counterclockwise. Genes belonging to different functional groups are color-coded.

426

Dashed area in the inner circle indicates the GC content of the chloroplast genome; blue

427

triangles indicate the positions of 14 markers (gcpm1–gcpm14) on the chloroplast genomes.

428

Fig. 2. Repeat structure analysis of the five Panax species chloroplast genomes.

429

(A) Histogram showing the frequency of repeats by length in the five Panax chloroplast

430

genomes.

431

(B) Histogram showing the number of four repeat types in each Panax chloroplast genome.

432

(C) Location of the 235 repeats on the five Panax species chloroplast genomes.

433

(D) Venn diagram showing the repeats shared among the five Panax species.

434

Fig. 3. Comparison of chloroplast genome sequences of five Panax species.

435

Pair-wise comparison of chloroplast genomes between Panax species using the mVISTA

436

program with P. ginseng cv. CP as the reference. Genome regions are color-coded as protein

437

coding (purple), rRNA or tRNA coding genes (blue), and noncoding sequences (pink).

438

Fig. 4. Validation of 14 molecular markers derived from InDel regions of five Panax

439

chloroplast genomes.

440

Schematic diagrams indicate InDel regions between Panax species. Tandem repeats and

441

inserted sequences are designated by pentagons and diamonds, respectively. Dotted and solid

442

lines indicate deleted and conserved sequences; left and right black arrows indicate forward

443

and reverse primers, respectively. The 14 InDel markers are denoted gcpm1 to gcpm14.

444

Different alleles are shown via capillary electrophoresis (A – F, I, N), and agarose gel

445

electrophoresis (G, H, K – M, O). Abbreviated species names shown on schematic diagrams 20 ACS Paragon Plus Environment

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446

and amplicons: PgCP, P. ginseng cv. CP; PgYP, P. ginseng cv. YP; Pq, P. quinquefolius; Pn,

447

P. notoginseng; Pj, P. japonicus; Pv, P. vietnamensis; M, 100-bp DNA ladder.



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Table 1. SSRs in the five Panax chloroplast genomes. Number of SSRs Motif

Repeat units

PgCP

Pq

Pn

Pj

Pv

A/T

14

16

19

17

17

C/G

4

3

1

7

3

AT/AT

6

7

6

7

6

AAG/CTT

1

1

1

1

1

AAT/ATT

1

1

3

2

2

AGC/CTG

1

1

-

-

-

AAAG/CTTT

3

3

3

3

3

AAAT/ATTT

2

2

2

2

4

AATT/AATT

1

1

1

1

1

ACCT/AGGT

2

2

2

2

2

AATCT/AGATT

2

2

2

2

2

AAAAT/ATTTT

-

-

1

1

-

AGATAT/ATATCT

-

-

-

-

1

ACTATG/AGTCAT

1

1

1

-

-

38

40

42

45

42

Mononucleotide Dinucleotide

Trinucleotide

Tetranucleotide

Pentanucleotide

Hexanucleotide Total SSRs

Abbreviations: PgCP, P. ginseng cv. CP; Pq, P. quinquefolius; Pn, P. notoginseng; Pj, P. japonicus; Pv, P. vietnamensis; SSR, simple sequence repeat.


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Table 2. Numbers and ratios of nucleotide substitutions and InDels among chloroplast genomes of five Panax species. PgCP

PgYP

Pq

Pn

Pj

Pv

PgCP

/

1

141

480

511

559

PgYP

3 (0.33)

/

142

481

512

560

Pq

34 (4.15)

35 (4.06)

/

509

508

542

Pn

101 (4.75)

98 (4.91)

110 (4.63)

/

484

548

Pj

92 (5.55)

93 (5.51)

90 (5.64)

111 (4.36)

/

331

102 (5.48) 103 (5.44) 104 (5.21) 124 (4.42) 74 (4.47) / Pv Note: The upper triangle shows the total nucleotide substitutions, while the lower triangle indicates the number of InDels. Ratios of nucleotide substitutions to InDels (S/I) are given in brackets. Abbreviations: PgCP, Panax ginseng cv. CP; PgYP, P. ginseng cv. YP; Pq, P. quinquefolius; Pn, P. notoginseng; Pj, P. japonicas; Pv, P. vietnamensis.



Table 3. Molecular markers developed to authenticate Panax species. Melting Expected PCR product size (bp) temperature PgCP PgYP Pq Pn Pj Pv (°C) a F: TGGGGGAATAAATAAGGAAGAA 54.7 gcpm1 392 392 393 391 416 430 R: TTAAAGAAGGCGGAGGTTTTTa 54.0 b F: TGGAAAGGCTGTTGTCACTG 53.2 gcpm2 194 194 161 160 161 161 R: TGCCAAGATTGCAGAAAGATTb 53.8 F: TGGTCAATCGCTGAAGAAAAa 53.2 gcpm3 449 449 449 474 449 449 R: CCGCGGCTTATATAGGTGAAa 57.3 F: CTCTTCCAAATTGATGTTCCAAa 56.6 gcpm4 295 295 318 295 295 295 R: TCCATGATACACCAGAACAATCAa 59.3 F: TCCTGAACCACTAGACGATGGa 53.3 gcpm5 457 457 457 465 469 444 R: TTTCGATAACTTCTTGATCCCTCTa 54.3 F: CTCGCACTAAGCTCGGAAATa 57.3 475 475 475 475 561 477 gcpm6 R: ACCATGGCGTTACTCTACCGa 53.9 F: TGGTGTGTTGAATCCACAATa 53.2 gcpm7 297 297 289 322 322 322 R: CCCCCTTTCCAATAATATCCAa 55.9 a F: TCCAAAGAGGAATCCTTCCA 53.3 gcpm8 237 237 237 313 237 237 R: CCAGCCTCTACTGGGGGTTAa 55.3 F: TCCAGGACTTCGAAAGGGTAa 53.4 gcpm9 492 492 492 492 486 511 R: ACACGATACCAAGGCAAACCa 53.7 F: CCGCTGTTATCCGCTACATTa 54.1 gcpm10 227 227 228 257 225 228 R: TCGTCTAAAATGCCTATACGAACTCa 54.9 F: CTGGACGATCCTATTTGGTCAb 53.7 gcpm11 220 220 220 238 220 202 R: TCCAGCTCCGTATGAAGGTCb 53.8 b F: GCAGAATACCGTCACCCATT 53.6 gcpm12 316 373 259 373 259 202 R: ATTTTGTCCGGATCCTCCTTb 53.8 F: AAGATTTTTATGAAGATACCGAAAATa 52.5 gcpm13 484 484 466 461 456 465 R: CGGTCAAATTCGAGGAAAGAa 55.3 F: TGGTTAGTTTCACCGGATTCAb 54.2 208 208 208 208 208 178 gcpm14 R: TTTTTGAGCCCATTTTTAAGGAb 54.6 Abbreviations: PCR, polymerase chain reaction; PgCP, Panax ginseng cv. CP; PgYP, P. ginseng cv. YP; Pq, P. quinquefolius; Pn, P. notoginseng; Pj, P. japonicus; Pv, P. vietnamensis. a Primers developed in our previous study17, and bprimers developed in this study Marker name

Forward (F)/reverse (R) primers


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Table 4. Marker combinations for each Panax species. Marker

Positions Regions CDS/IGS

PgCP

Allele types for each species PgYP Pq Pn Pj

Pv

trnK-UUU–rps16 IGS C C C D B A rps16–trnQ-UUG IGS A A B B B B atpH-atpI IGS B B B A B B atpH-atpI IGS B B A B B B trnE-trnT IGS B B B B A C trnE-trnT IGS B B B B A B rbcL-accD IGS B B C A A A petA-psbJ IGS B B B A B B clpP-psbB IGS B B B B B A rpl14–rpl16 IGS B B B A B B ycf2 CDS B B B A B C ycf1 CDS B A C AD C D ndhF–rpl32 IGS A A B C D C ycf1 CDS A A A A A B Abbreviations: CDS, coding sequences; IGS, intergenic spaces; PgCP, P. ginseng cv. CP; PgYP, P. ginseng cv. YP; Pq, P. quinquefolius; Pn, P. notoginseng; Pj, P. japonicus; Pv, P. vietnamensis. A, B, C, and D refer to different allele types.

gcpm1 gcpm2 gcpm3 gcpm4 gcpm5 gcpm6 gcpm7 gcpm8 gcpm9 gcpm10 gcpm11 gcpm12 gcpm13 gcpm14



Figure 1


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Figure 2



Figure 3


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Figure 4



Table of Contents Graphic


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Authentication Markers for Five Major Panax Species Developed via

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